| ObjectiveAcute respiratory distress syndrome(ARDS)has a high mortality and disability rate in clinical practice.At present,the mortality rate in most studies is as high as 30%～40%.In the past 50 years,randomized clinical trials have optimized ARDS management strategies such as lung protective mechanical ventilation and fluid therapy,and shortened the time of mechanical ventilation and organ failure.Due to the lack of effective treatment drugs,the mortality of patients with ARDS has not been significantly reduced,and the sequelae of the decline in the quality of life caused by the lung fiber of the surviving patients are increasingly prominent.Especially in the global pandemic of COVID-19,even though COVID-19 patients have survived the early acute inflammatory factor storm after they developed into ARDS,pulmonary fibrosis that began within 24 hours of diagnosis of ARDS seriously affects the prognosis and long-term quality of life of patients,and increases the social burden.Multi-center studies have found that the benefits of using methylprednisolone to suppress inflammation in the acute phase are mainly reflected in the treatment of ARDS patients with high inflammatory response,and long-term use of methylprednisolone will increase the mortality rate,which indicates that there are other important cell damage mechanisms of non-inflammatory response in the pathogenesis of ARDS.Animal experiments have confirmed that oxidative stress response is one of the main mechanisms of pulmonary fibrosis after lung injury,and the nuclear factor erythrocyteE2-related factor-2(Nrf2)signal pathway is the most important antioxidant stress pathway in cells.The lung tissue of ARDS patients exceeds the metabolic capacity of the body under continuous stress.The insufficient resistance of the antioxidant stress system leads to the continuous production of reactive oxygen species,and the lung epithelial cells will suffer oxidative stress damage repeatedly.The excessive and inappropriate repair of many activated lung fibroblast effector cells under continuous injury,accompanied by the secretion of a large amount of collagen and deposition in the extracellular matrix,eventually led to the irreversible occurrence of pulmonary fibrosis.Therefore,it is an important direction to treat pulmonary fibrosis after ALI by effectively interfering with the upregulation of Nrf2 antioxidant signal pathway to reduce oxidative stress response damage.Melatonin is a natural antioxidant secreted in the body and could regulate the circadian rhythm.Many previous studies have proved that it can significantly activate Nrf2 antioxidant stress signal transduction,and up-regulate the activities of downstream antioxidant enzymes and detoxification enzymes,reducing oxidative stress reactions.Some scholars have found that melatonin has a significant inhibitory effect on the occurrence and progression of pulmonary fibrosis,but the specific molecular mechanism is not completely clear.Mitochondria,as the main site of active oxygen generation in the body,its autophagy level is closely related to the level of active oxygen in the body.Whether melatonin can regulate mitochondrial autophagy and reduce oxidative stress response to improve pulmonary fibrosis after acute lung injury is currently lack of relevant research.This subject mainly studied the fibroproliferative reaction of the lung in the early stage of ARDS patients under the stimulation of oxidative stress,and the application of safe dose of melatonin can interfere with Nrf2-related signal pathway,improve the oxidative stress response in acute lung injury by enhancing mitochondrial autophagy,and inhibit the occurrence and progress of pulmonary fibrosis after acute lung injury.MethodsIn this clinical study,patients with moderate and severe ARDS who meet the Berlin definition criteria were included as the experimental group,and patients with respiratory failure due to nonARDS factors in the same period who need mechanical ventilation were included as the control group.The clinical data of patients were collected,and bronchoalveolar lavage and chest CT scanning were performed on the first and seventh days after the diagnosis after the immediate family members agreed and signed the informed consent.Collect BALF to detect fibrosis-related markers(NT-PCP-III,TGF– β1),oxidative stress indicators(MDA,SOD),and inflammatory factors(TNF-α、 IL-1β)The fibrosis score of the chest CT at the corresponding time point.We analyzed the differences in clinical indicators,fibrosis indicators,inflammatory factors,and oxidative stress levels between two groups of patients at the same time point,and the subgroup analysis of the death group and the survival group of the ARDS group was further carried out to preliminarily clarify that oxidative stress reaction participated in the early fibroproliferative reaction of acute lung injury and affected the outcomes of patients.In vitro study,we cultured human bronchial epithelial 16 HBE cell line,observed the morphological changes of epithelial-mesenchymal transformation of 16 HBE cells stimulated by LPS at the living cell workstation,observed the mitochondrial changes of EMT cell model and the therapeutic effect of melatonin by transmission electron microscopy,and observed the role of Nrf2/SQSTM1/P62 channel pathway in it by immunoblotting and immunofluorescence,as well as the regulatory effect of melatonin on Nrf2/SQSTM1/P62 channel pathway.Finally,we used lentivirus transfection to knock down Nrf2 gene and inhibitor XRK3F2 to inhibit P62 respectively to further explore the regulatory effect of melatonin on Nrf2/SQSTM1/P62 channel pathway and the mechanism of action on mitochondrial autophagy and pulmonary epithelial cell EMT in the process of pulmonary fibrosis after acute lung injury.ResultsClinical research section:(1)During the study period,64 patients with ARDS were ultimately included,and 57 patients in the control group who did not require mechanical ventilation for ARDS.Compared to patients in the non ARDS group,patients in the ARDS group had lower oxygenation index and higher mortality rate;(2)Within 24 hours of admission,the inflammatory response,oxidative stress levels,and fibroproliferative indicators in the ARDS group were significantly higher than those in non ARDS patients during the same period of admission,and there were still high levels of oxidative stress in the BALF of ARDS group patients on the 7th day;(3)There is a correlation between the concentration of NT-PCP-III in BALF and the level of oxidative stress in ARDS patients;(4)Compared with surviving patients,non-surviving patients in the ARDS group showed a significant increase in oxidative stress levels in BALF after 24 hours of admission.In vitro research section:(1)LPS induced the morphological changes of EMT in 16 HBE cells,accompanied by the increase of total reactive oxygen species and mitochondrial reactive oxygen species;(2)Melatonin can up-regulate the expression level of Nrf2 and P62 in 16 HBE cells,and then enhance mitochondrial autophagy,reduce oxidative stress reaction,and inhibit the EMT process of16 HBE cells induced by LPS.(3)The protective effect of melatonin can be eliminated by the lentivirus targeted knockdown of Nrf2 or(and)XRK3F2 inhibitor inhibiting P62.ConclusionsOxidative stress is involved in the early pulmonary fibroproliferative reaction of ALI/ARDS patients and affects the prognosis of patients;Melatonin enhances mitochondrial autophagy by regulating the Nrf2/SQSTM1/P62 signal pathway,reduces the oxidative stress response of the lung after acute lung injury,inhibits the process of LPS-induced pulmonary epithelial EMT,and finally improves the occurrence of pulmonary fibrosis after acute lung injury. |
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